Immunoassays for high positively charged proteins

ABSTRACT

The present invention relates to immunoassays for quantification of a high positively charged protein, such as a FGF-18 protein, in human synovial fluid sample.

FIELD OF THE INVENTION

The present invention relates to immunoassays for evaluating affinity ofan antibody for FGF-18 protein and for quantification of FGF-18 proteinin human synovial fluid sample.

BACKGROUND OF THE INVENTION

Immunoassays are biochemical tests that exploit the ability ofantibodies to specifically bind a molecule among a complex mixture (e.g.biological matrix). This ability can be used to detect or quantify therecognized molecule, the so-called analyte, or vice versa, i.e. theantigen can be used to capture and allow quantification of a specificantibody. The binding event is associated to the generation of ameasurable signal, which is usually compared to the signal generated bya reference sample at a known concentration. The first immunoassays wereall based on the labelling of antibodies with radioactive iodine(RadioImmunoAssays, RIA). In the '60s there were the first experimentswith enzyme based immunoassays (EnzymeImmunoAssays EIA, or Enzyme LinkedImmunoSorbent Assay, ELISA), that have become the most popular duringthe years. Nowadays there are several kinds of labels that can be used,from enzymes to fluorescent probes, DNA, etc. Moreover unlabelledreagents can be used.

Different immunoassays technologies are currently used, such asAlphaLISA Technology, Gyrolab Technology and Imperacer Technology.

The Gyrolab immunoassay platform is a microfluidics-based automatedsystem, composed by a liquid handling system, a fluorescence detectionsystem and compact disc (CD) microlaboratory. The immunoassay isperformed on affinity capture columns embedded in microstructures intothe CD. There are different kinds of CDs, allowing the processing ofdifferent analyte volumes, in order to have different performances interms of dynamic range, sensitivity and type of assay. In a CD there arefrom 12 to 14 segments, each segment is made up by 8 microstructures.Each microstructure contains a complex system of nanofluidics, thatallows the passage of reagents, wash buffers and analytes, and ends withthe affinity column, prepacked with streptavidine beads. The immunoassaytakes place by using the centrifugal force generated by the CD rotation:the common reagents are dispensed by the liquid handling system in acommon channel (one for each segment) while samples are deposited in anindividual inlet to avoid cross-contamination. A slow rotation of the CDallows the volume definition by eliminating the extra-volume using theoverflow channel: a hydrophobic barrier prevents the passage of thefluid to the column. After volume definition a faster rotation of the CDallows the breakage of the hydrophobic barrier, and the liquid can flowthrough the column. The first reagent must be labelled with biotin, inorder to bind to the streptavidine beads. At the end of the immunoassay(for example a secondary Ab in a sandwich immunoassay) a reagentlabelled with a fluorescence probe is added. A laser inside theworkstation is able to excite the fluorophore and then the fluorescenceresponse is read. Between the different steps of reagents addition thereare several washing steps that make the immunoassay more specific. Theinstrument has pre-determined protocols, but many parameters can bevaried (composition of the wash buffers, number of washing steps, lengthof CD spinning) to optimize a method in order to obtain the bestresults. Different immunoassay formats (sandwich, direct, indirect,competitive and bridging) can be set up. The only requirement is theavailability of a biotinilated and a fluorescence probe labelledreagent.

SUMMARY OF THE INVENTION

In one aspect the invention provides a method for pre-treating a humansynovial fluid sample for immunoassay comprising

-   -   adding hyaluronidase solution to the human synovial fluid        sample,    -   incubating said sample at room temperature (RT),    -   centrifuging the human synovial fluid sample.

In another aspect the invention provides a method for quantification ofa high positively charged protein in a human synovial fluid samplecomprising the steps of

-   -   a) pre-treating the human synovial fluid sample, the        pre-treating step comprising        -   adding hyaluronidase solution to the human synovial fluid            sample,        -   incubating said sample at room temperature (RT),        -   centrifuging the human synovial fluid sample,    -   b) diluting the pre-treated human synovial fluid sample with a        buffer,    -   c) immobilizing a biotinylated antibody against the high        positively charged protein to a column,    -   d) washing the column to remove unbound antibody with a standard        wash buffer,    -   e) contacting in the column the pre-treated and diluted human        synovial fluid sample with the immobilized biotinylated antibody        under conditions in which the antibody binds specifically to the        high positively charged protein, to produce an antibody-protein        complex,    -   f) washing the column complex with a standard wash buffer;    -   g) adding to the antibody-protein complex in the column a        fluorescent dye labelled antibody specific for the high        positively charged protein to produce a measurable response, and        washing the column with a standard wash buffer,    -   h) measuring the response produced,    -   i) determining a quantity of high positively charged protein in        the sample by comparing the    -   response produced with the sample to the response produced with        a calibration standard.

In a further aspect the invention provides a method for automaticquantification of a high positively charged protein in a human synovialfluid sample comprising the steps of

-   -   a) pre-treating the human synovial fluid sample, the        pre-treating step comprising        -   adding hyaluronidase solution to the human synovial fluid            sample,        -   incubating said sample at room temperature (RT),        -   centrifuging the human synovial fluid sample,    -   b) diluting the pre-treated human synovial fluid sample with a        buffer,    -   c) immobilizing a biotinylated antibody against the high        positively charged protein to a column,    -   d) washing the column to remove unbound antibody with a standard        wash buffer,    -   e) providing a injection means for automatic transfer of the        pre-treated and diluted human synovial fluid sample to the        column,    -   f) washing the injection means with a high ionic force buffer        before the pre-treated and diluted human synovial fluid sample        transferring to the column,    -   g) transferring the pre-treated and diluted human synovial fluid        sample to the column, thereby contacting the pre-treated and        diluted human synovial fluid sample with the immobilized        biotinylated antibody under conditions in which the antibody        binds specifically to the high positively charged protein, to        produce an antibody-protein complex,    -   h) washing the injection means with a high ionic force buffer        after the step g)    -   i) washing the column with a standard wash buffer;    -   j) adding to the antibody-protein complex in the column a        fluorescent dye labelled antibody specific for the high        positively charged protein to produce a measurable response, and        washing the column a standard wash buffer,    -   k) measuring the response produced,    -   l) determining a quantity of the high positively charged protein        in the sample by comparing the response produced with the sample        to the response produced with a calibration standard.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows reaction of Sulfo-NHS-LC-Biotin with primary amine. Notethat NHS is a leaving group (byproduct) in the reaction. The leavinggroup and any non-reacted biotin reagent are removed during thedesalting step.

FIG. 2 shows graph of the log concentration of analyte vs RLUs obtainedafter combined incubation with alexa-647 labelled F05. Mean results ofthree replicates, error bars show SEM.

FIG. 3 shows graph of the log concentration of analyte vs RLUs obtainedafter combined incubation with alexa-647 labelled F44. Mean results ofthree replicates, error bars show SEM.

FIG. 4 shows graph of the log concentration of analyte vs average RLUsobtained after combined incubation with alexa-647 labelled mAb F05 in 4independent experiments. Mean results of three replicates.

FIG. 5 shows graph of the log concentration of analyte vs average RLUsobtained after combined incubation with alexa-647 labelled F44 in 4independent experiments. Mean results of three replicates.

FIG. 6 shows analysis of binding differences between F44 and F05. (A)scatterplot of all ˜4000 peptides within the CLIPS discontinuous matrixdataset. For each peptide, the ELISA value obtained for F05 and F44 wasplotted as an XYscatter. Red line; 45° reference. Green line; LOESSspline fit. One data point was coloured magenta to trace over figuresA-C. (B) LOESS normalized data obtained from fig A. (C) MA-plot offigure B. Datapoints above or below 0.5 and −0.5 are scored andcoloured. (D) Frequency table of peptides scored in C. blue linesindicated higher binding for F44. Yellow lines indicate higher bindingfor F05.

FIG. 7 shows visualization of binding areas onto structure of ahomologue protein. 21-27 is coloured green. 7-21 is coloured yellow.153-164 is coloured blue.

FIG. 8 shows graph of the log concentration of analyte vs average RLUs.Mean results of three replicates, error bars show SEM.

FIG. 9 shows histogram representing the result of BCC of spiked samples(SS) and Synovial fluid blank samples (SF) with various MRD. Average of3 replicates, error bars represent SEM.

FIG. 10 shows histogram representing the result of mean RLU of QCsamples prepared in Rexxip HN at three level of concentration (QC-Low=30pg/mL, QC-Medium=500 pg/mL, QC-High=15000 pg/mL) With (treated) orwithout (untreated) hyaluronidase digestion. Average of 3 replicates,error bars represent SEM.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Thepublications and applications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

In the case of conflict, the present specification, includingdefinitions, will control. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of skill in art to which the subject matter hereinbelongs. As used herein, the following definitions are supplied in orderto facilitate the understanding of the present invention.

The term “comprise” is generally used in the sense of include, that isto say permitting the presence of one or more features or components.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise.

The term “FGF-18 compound”, “FGF-18 protein” or “FGF-18”, as usedherein, is intended to be a protein maintaining at least one biologicalactivity of the human FGF-18 protein. FGF-18 may be native, in itsmature form, a recombinant form or a truncated form thereof. Biologicalactivities of the human FGF-18 protein include notably the increase inchondrocyte or osteoblast proliferation (see WO98/16644) or in cartilageformation (see WO2008/023063). Native, or wild-type, human FGF-18 is aprotein expressed by chondrocytes of articular cartilage. Human FGF-18was first designated zFGF-5 and is fully described in WO98/16644. SEQ IDNO:1 corresponds to the amino acid sequence of the native human FGF-18,with a signal peptide consisting of amino acid residues 1(Met) to27(Ala). The mature form of human FGF-18 corresponds to the amino acidsequence from residue 28(Glu) to residue 207(Ala) of SEQ ID NO: 1 (180amino acids). FGF-18, in the present invention, may be produced byrecombinant method, such as taught by the application WO2006/063362.Depending on the expression systems and conditions, FGF-18 in thepresent invention is expressed in a recombinant host cell with astarting Methionine (Met) residue or with a signal sequence forsecretion. When expressed in prokaryotic host, such as in E. coli,FGF-18 contains an additional Met residue in N-terminal of its sequence.For instance, the amino acid sequence of human FGF-18, when expressed inE. coli, starts with a Met residue in N-term (position 1) followed byresidues 28 (Glu) to residue 207 (Ala) of SEQ ID NO: 1.

The term “truncated form” of FGF18, as used herein, refers to a proteinwhich comprises or consists of residues 28(Glu) to 196(Lys) of SEQ IDNO: 1. Preferably, the truncated form of FGF-18 protein is thepolypeptide designated “trFGF-18” (170 amino acids; also known asrhFGF18 or sprifermin), which starts with a Met residue (in N-terminal)followed by amino acid residues 28 (Glu)-196 (Lys) of the wild-typehuman FGF-18. The amino acid sequence of trFGF-18 is shown in SEQ IDNO:2 (amino acid residues 2 to 170 of SEQ ID NO:2 correspond to aminoacid residues 28 to 196 of SEQ ID NO:1). trFGF-18 is a recombinanttruncated form of human FGF-18, produced in E. coli (see WO2006/063362).trFGF-18 has been shown to display similar activities as the maturehuman FGF-18, e.g. it increases chondrocyte proliferation and cartilagedeposition leading to repair and reconstruction for a variety ofcartilaginous tissues (see WO2008/023063).

The term “positively charged” refers to protein that contain morepositively charged basic amino acids (such as lysine, arginine andhistidine) than negatively charged acidic amino acids and/or are foldedso that positively charged amino acids are exposed on their surfaceand/or exhibit an overall positive charge at the pH of the medium (whenthe pH of said medium is lower than the pI, ie isoelectric point). Theseproteins are usually basic (pI usually higher than 7). The term “highpositively charged protein” refers to proteins being very basic, i.ehaving a high pI, preferably at about or above 9.5. Lysozyme, FGF-2 andFGF-18 for instance, are basic proteins, highly positively charged. Thisterm is well within the knowledge of the skilled person.

The term “room temperature” refers to a temperature usually in the rangeof at or about 15 to 25° C., such as at or about 15° C., 18° C., 20° C.,22° C. or 25° C.

According to the project phase different types of methods are needed,for example in the early phases only a quick and dirty evaluation isrequired, or a method can be qualified. For GLP and clinical studiesmethods need to be validated. Moreover not in every case the evaluationof immunogenicity is required.

Measurement of drug concentrations (PK) and anti-drug antibody (ADA) inbiological matrices is an important aspect of drug development. Theresults of toxicokinetic, pharmacokinetic and bioequivalence studies areused to make critical decisions supporting the safety and efficacy of amedicinal drug. It is therefore vital that the applied bioanalyticalmethods used are well characterized, fully validated and documented to asatisfactory standard in order to yield reliable results. Non-clinical(pharmaco-toxicological) studies submitted in a marketing authorizationapplication must be carried out in conformity with the Good LaboratoryPractice. Methods used in non-GLP pre-clinical studies should bequalified but not necessarily developed in a GLP facility and fullyvalidated. Human bioanalytical studies fall outside of the scope of GLP,as defined in Directive 2004/10/EC, while the principles of GoodClinical Practice (GCP) should be followed. The regulatory authorities(FDA, EMA) issued official guidelines describing validation of PK andADA assay that must be followed in order to assure reliability of thedata produced using a validated method. Moreover, white papers writtenby the scientific community are covering new topics that arise in thefield between different editions of the official guidelines. An assaylife cycle can be categorized into 3 general phases: method development,pre-study validation, and in-study validation. During methoddevelopment, an assay concept is evaluated, which will be confirmedduring the pre-study validation phase, and applied during the in-studyvalidation phase. The pre-study validation phase takes place after themethod optimization is concluded and the assay is considered performant:this means that the critical assay reagents should be identified andproduced if necessary. Based on the available reagents the mostappropriate technology is chosen, the assay format and the batch sizeare defined. The Minimum Required Dilution (MRD) is selected to minimizematrix effect and the method range is defined and sensitivity isconfirmed. Standard curve concentrations and a regression model forfitting a curve to calibration data should be established during methoddevelopment. Before starting with the experimental phase a validationplan must be produced. The documentation should include a description ofthe intended use of the method and a summary of the performanceparameters to be validated, a summary of the proposed experiments andthe target acceptance criteria for each performance parameter evaluated.After completion of the validation exercise, a comprehensive report mustbe produced. The report summarizes the assay performance results and anyother relevant information related to the conditions under which theassay can be used without infringing the acceptance criteria. The maincharacteristics of a bioanalytical method that are essential to ensurethe acceptability of the performance and the reliability of analyticalresults are: selectivity, lower limit of quantitation, method range, theresponse function (calibration curve performance), accuracy, precision,matrix effects, stability of the analyte in the biological matrix andthe stock and working solutions under the entire period of storage andprocessing conditions.

Immunoassays for the quantification of drug or anti-drug antibodies relyclosely on the quality of the antibodies used. This means that one canuse different technologies and formats, but the starting point mustalways be a good antibody pair. The role of antibodies in an immunoassaygenerally includes capture and detection reagents for PK assays andpositive controls for immunogenicity assays. When several types of assayformats and reagents are considered for the same application, it isadvisable to initiate the generation of the different types of reagentsin parallel, increasing the likelihood of obtaining optimal reagentswith the appropriate and pre-specified characteristics. Conducting anappropriate risk assessment provides an opportunity to adequatelybalance resources and timelines. The best strategy is to consider theobjectives of the study in question, the stage of the drug developmentprogram, potential assay formats, species of origin and type of matricesof samples to be tested, required assay sensitivity and specificity, aswell as the potential need for reagent reactivity to multiple analytes.Examples of useful information to be collected include: (a) therequirements for sensitivity as well as specific and selectiveinteractions between a reagent and the target analyte; (b) the need toreduce potential non-specific interactions with other assay components,such as heterophilic antibodies and their interactions in PK assays (c)the need to reduce potential cross-reactivity in the target samplepopulation (e.g., rheumatoid factor); (d) the need for discriminationbetween analyte isoforms and its proteolytic byproducts; (e) therequirement for detecting free analytes versus those in largercomplexes; and (f) detecting anti-drug antibodies bound to thetherapeutic with or without treatment of the sample with acid. Eachassay requires different antibodies types: for examples during early MAbtherapeutic discovery programs, generic anti-IgG reagents may be used,which may later become less desirable as target-specific reagents aregenerated.

Whatever the technology used, without a good raw material, especiallyantibodies, the goodness of the assay can be impacted. For this reason,to better select the antibodies of interest, affinity determinationusing Gyrolab and epitope mapping using the CLIPS technology wereexplored. The evaluation of the new technologies was conducted takinginto consideration the phase of the project trying to implement the goodplatform to use based on the specific needs (e.g. required sensitivity,flexibility, ease of transfer).

Gyrolab was chosen to develop a PK method to quantify a highlypositively charged drug, such as a FGF-18 protein, preferablysprifermin, in human synovial fluid. Two difficulties were encounteredduring method development: high isoelectrical point of the analyte (adrug) and high viscosity of the matrix (the sample). The challenge wasto find a way (type of buffer for analytic dilution, washing procedure,etc) to help reducing issues related to sickness of the molecule and asample pre-treatment able to fluidify the matrix without compromisingthe analyte stability.

Determination of antibodies affinity can have a great value for a bettercomprehension of the best format to be used, or to understand possiblepitfalls when developing new immunoassays.

In a biological interaction, there are two partners: the one with thelower molecular weight is usually called the ligand (L), and themacromolecular binding partner is called the receptor (R). In theimmunocomplexes, the antibody is considered as the receptor, while theanalyte is considered the ligand. In solution, the total concentrationof a receptor is made up by the fraction of receptor free and theligand-bound (assuming that the receptor has a single binding site forthe ligand, so that any molecule is either free or bound). As well, anyligand molecule must be either free or bound to a receptor molecule.This leads to these mass conservation equations:

[R]=[RL]+[R] _(f)

[L]=[RL]+[L] _(f)

Where [R] and [L] are total concentrations of receptor and ligandrespectively, [R]_(f) and [L]_(f) are the free concentrations of the twomolecules, and [RL] is the concentration of the receptor-ligand complex.

In this system, there will be a continuous passage of molecules from thefree to the bound state.

${R + L}\overset{Kon}{\leftarrow}{\underset{Koff}{\rightarrow}{RL}}$

Where the k_(on) is the second-order rate constant for complexassociation and the K_(off) is the first-order rate constant for complexdissociation.

When the rate of association and dissociation of the complex become tobe equal, the equilibrium is reached. The position of this equilibriumis quantified in terms of dissociation constant K_(d):

$K_{d} = {\frac{{\lbrack R\rbrack_{f}\lbrack L\rbrack}_{f}}{\lbrack{RL}\rbrack} = {\frac{1}{K_{a}} = \frac{K_{off}}{K_{on}}}}$

The relative affinities of different receptor-ligand complexes areinversely proportional to their K_(d) values, so the strength of bindingto the same molecule can be compared using the K_(d) value for differentbinding partners. The association constant (K_(a)) is the inverse of theK_(d).

In most of the cases, the affinity between a receptor and a ligand issuch that a large excess of ligand is required to effect significantbinding to the receptor; thus under most experimental conditions, theformation of the binary complex proceeds with little change in theconcentration of free ligand. Thus the association reaction proceedswith pseudo-first-order kinetics:

[RL] _(t) =[RL] _(eq)[1−exp(−k _(obs) t)

Where [RL]_(t) is the concentration of the binary complex RL at the timet, [RL]_(eq) is the concentration of the binary complex at theequilibrium, and k_(obs) is the experimentally determined value for thepseudo-first-order rate constant for approach to equilibrium.

For reversible binding, the value of Kobs is directly proportional tothe concentration of the ligand:

k _(obs) =k _(off) +k _(on) [L] _(f)

Therefore, one can determine the value of k_(obs) at different ligandconcentrations. By plotting k_(obs) as function of ligand concentrationa linear fit with slope equal to k_(on) and intercept equal to k_(off)is obtained.

The most common and easy way to study receptor-ligand interactions is towait after equilibrium has been established, since these kineticsusually occur in a very short time.

At the equilibrium the concentration of the RL complex is constant, andthe rate of complex association and dissociation is equal.

${K_{d} = {\frac{{\lbrack R\rbrack_{f}\lbrack L\rbrack}_{f}}{\lbrack{RL}\rbrack} = {{\frac{K_{off}}{K_{on}}\mspace{45mu} \frac{d\lbrack{RL}\rbrack}{dt}} = {{k_{on}\lbrack R\rbrack}_{f}\lbrack L\rbrack}_{f}}}},{\frac{- {d\lbrack{RL}\rbrack}}{dt} = {{{k_{off}\lbrack{RL}\rbrack}\mspace{45mu} {{k_{on}\lbrack R\rbrack}_{f}\lbrack L\rbrack}_{f}} = {k_{off}\lbrack{RL}\rbrack}}},{\lbrack{RL}\rbrack = \begin{matrix}\frac{K_{on}}{K_{off}} & {\lbrack R\rbrack_{f}\lbrack L\rbrack}_{f}\end{matrix}}$

Considering that k_(on)/k_(off) is equivalent to the associationconstant K_(a):

[RL]=K _(a) [R] _(f) [L] _(f)

There are various techniques that are currently used for the affinitymeasurement; the most employed in literature are Isothermal TitrationCalorimetry (ITC), Surface Plasmon Resonance (SPR) and KinExA.

Gyrolab technology was used to evaluate the affinity of an antibody forits antigen. This technology is based on the competition of theimmobilized antigen for the binding to the antibody of interest withincreasing amounts of free antigen at the equilibrium. Only the antibodynot bound to the free antigen in solution is captured by the immobilizedantigen, allowing the determination of its concentration using afluorophore (i.e. a fluorescent dye) for the detection. According to thedeveloped procedure, the steps required for the determination of theaffinity are:

-   -   Biotinilation of the antigen and labeling of the antibody with a        fluorescent dye, for instance Alexa-647,    -   Investigations on the capture conditions,    -   Determination of the fixed Ab concentration,    -   Determination of the binding equilibrium time,    -   Confirmation of the obtained KD: repetitions and variation in Ab        concentration.

Since the labelling of the reagents is a crucial step, the capturereagent (sprifermin) was labelled with different ratios of biotin andused to capture the antibody in comparison with the unlabelled antigen.Sprifermin is a highly positively charged protein, therefore a highaspecific binding of the analyte to the column (for example CD column ofGyrolab technology) was observed. In order to reduce this effect, amodified method with more stringent washing steps was employedsuccessfully. The chosen capturing reagent was sprifermin labelled with1:2 to 1:20 ratio of biotin (such as 1:2, 1:10 or 1:20), preferably 1:5to 1:15 ratio of biotin or even preferably 1:10 ratio of biotin. Oncethe capture reagent was established (sprifermin labelled preferably at1:10), it was used to determine the fixed antibody (Ab) concentration: astandard curve of both the antibodies under evaluation was analysedwithout pre-mixing them with the free antigen. The choice of the bestconcentration was based on two factors: the chosen concentration musthave lied on the linear part of the curve and the response must havebeen enough high to allow enough room for signal reduction with theincrease of antigen concentration in the pre-mix. Two antibodies againstFGF-18 have been used: F05 and F44. Once labelled with the fluorescentdye, both labelled antibodies were used at a concentration ranged from0.1 pM to 100 nM, such as for instance 0.1 or 0.5 pM, 0.001, 0.005,0.01, 0.05, 0.1, 0.5, 1.5, 10, 50 or 100 nM. Preferably, theconcentration is ranged from 0.05 to 1 nM. The even preferredconcentration resulted to be at or about 0.1 nM. The two mAbs at theselected concentration were incubated with different amounts of antigen(spanning from 0.5 pM to 2 μM) for 1 hour (1 h) or 24 hour (24 h). Theresponses obtained after 1 h and 24 h of incubation were very similar,therefore the calculated K_(D) was comparable for the two mAbs. Arguingthat equilibrium was already reached at this time, all the followingexperiments were performed with a pre-mixing of 1 hour, a favourablecondition in order to speed up the entire development. These preliminarydata were also useful for the first K_(D) estimation. K_(D) of the F05mAb resulted to be around 1 nM, while the mAb F44 seemed to have higheraffinity with an estimated K_(D) of around 200 pM. To have a goodcalculation of the K_(D), a K_(D) controlled experiment was performedand the condition to be satisfied was: the ratio between fixed mAbconcentration and the calculated K_(D) should have been less or equalto 1. In both cases this requirement was fulfilled; the ratio for F05was around 0.09 and for F44 was around 0.2, abundantly below 1.

These experiments were confirmed by repeating 4 times the assessmentusing the developed procedure also changing the fixed mAb concentration.F05 gave robust results, with stable responses and conserving theestimated affinity constant around 1 nM for all the experimentsperformed in a KD controlled fashion. The F44 showed a higher affinitythan F05, being around 300 pM, but results were less reproducible,suggesting that this behaviour can be due to the reagents used: with ahigher affinity antibody the determination is more challenging.

The second aspect addressed to evaluate quality of the antibodies wasthe epitope mapping. Since these antibodies are used in a sandwichimmunoassay, the recognized epitopes can be a useful information notonly to select the best pair of antibodies but also for datainterpretation. By knowing the exact epitopes recognized by theantibody, it is possible to evaluate the ability of the assay tospecifically recognize the intact drug or digested products, informationvery useful for PK data interpretation during drug development. Epitopemapping was performed according to Pepscan Presto (The Neederland). Thistechnology was chosen for its ability to recognize not only linearepitopes as the most conventional techniques but also conformational anddiscontinues epitopes in a short time. The antigen (sprifermin) wasconverted in a library of around 4000 overlapping peptides that weretested as such and with single residue mutagenesis. Moreover they wereconformed in loops using the CLIPS technology: as such, with mutagenesisand finally with the juxtaposition of sequences coming from all theprotein length. All these sets of peptides were tested in aPepscan-based ELISA optimized ad hoc for the binding properties of theantibodies used. Data analysis was challenging because the twoantibodies showed very similar binding patterns, but since they are usedin a sandwich ELISA, there should necessarily be a difference in the twoepitopes. Therefore, the binding patterns were analysed in comparison byhighlighting differentially binding peptides. A common binding regionwas found (amino acid residues 21-27 of SEQ ID NO. 2). In addition asecond binding region (amino acid residues 7-21 of SEQ ID NO. 2 for F05and 153-164 of SEQ ID NO. 2 for F44) was found for both mAbs. Theseregions are respectively at the N-term and C-term domain of the proteinprimary sequence and, based on the tertiary structure of a protein withhigh homology of sprifermin, they should be in proximity of the regionof amino acid residues 21-27 of SEQ ID NO. 2.

Two antibodies, F05 and F44, were used to develop a PK assay to quantifysprifermin in human synovial fluid. Since the matrix showed a very highinterference and the volume of samples collected from patients could bevery limited, Gyrolab was chosen as preferred technology with respect tothe sandwich ELISA already validated to quantify the same drug in humanserum. The antibody couple was tested in both combinations of captureand detection, resulting that F44 could be used as capture reagent whileF05 as detection reagent. This behaviour confirms the major affinitymeasured for F44: in fact the most affine Ab is normally used to capturean analyte in a complex matrix.

An important aspect inventors had to take into account was the analyte.Sprifermin is a recombinant protein of around 20 kDa characterized by anisoelectrical point of 10.4. That means that at a neutral pH the proteinis positively charged. As a consequence, it is sticky and has thetendency to bind to the glass and plastic surfaces. Therefore, thebuffer for sample dilution was optimized to avoid loss of analyte duringsample handling. Different buffers have been tested: Rexxip HN, RexxipHN Max, Diluent buffer 1 and Diluent buffer 10. Among these buffers, theone with the best performances was the Rexxip HN (a buffer developed forpositively charged analytes).

The second issue related to this method development was the complexityof the matrix. Unspiked synovial fluid, as well as spiked with theanalyte, were diluted using different MRDs to find the optimal dilution.During the experiments conducted to find the best MRD, inventorsexperienced that synovial fluid is highly viscous and difficult to behandled. Moreover a strange behaviour in term of response in the assaywas observed: the lower dilution, the better result in term of CV % and% BIAS. For these reasons it was decided to improve the handling ofsynovial fluid by adding a pre-treatment procedure. Digestion withhyaluronidase was assessed, as synovial fluid is made in particular ofhyaluronic acid. Mild conditions in terms of temperature and durationwere applied to reduce impact of the treatment on analyte stability.After several trials, inventors realized that hyaluronidase digestion ofsynovial fluid alone was not enough to reduce sample viscosity; needleclotting, contamination of other samples, bad % CV and bad % BIAS wereobserved in many experiments performed varying hyaluronidaseconcentration and incubation time. Therefore, to improve the performanceof the method, a step of centrifugation was tested in combination withhyaluronidase digestion providing good results in terms of % BIAS and %CV without impacting the analyte stability. The final samplepre-treatment procedure was set up (centrifugation twice at 13'000 rpmfor 5 minutes and incubation for 30′ at RT in shaking with 20 μg/mL ofhyaluronidase) improving handling of the samples, increasing matrixfluidity and avoiding needle clotting phenomena. After havingestablished dilution buffer, MRD and sample pretreatment, to ensure thatthe analyte doesn't really bind to the instrument, needles carry-overassessment was performed. As expected, Sprifermin still showed highbinding to the Gyrolab needles (due to its high positive charge) even ifRexxip HN was used, impacting on low concentrated Spiked Sample (SS).Therefore the method was modified by adding additional washing stepswith a high salt solution before analyte addition step. The method thuscomprises at least 2 wash steps: one before analyte addition step andone after said analyte addition step.

In a further aspect the invention provides a method for pre-treating ahuman synovial fluid sample for immunoassay comprising

-   -   adding hyaluronidase solution to the human synovial fluid        sample,    -   incubating said sample at room temperature (RT)    -   centrifuging the human synovial fluid sample

The hyaluronidase concentration is ranged from 0.1 to 30 μg/mL, such as0.1, 1, 5, 10, 15, 20, 25 or 30 μg/mL. Preferably, it is ranged from 5to 20 μg/mL. Even preferably it is at or around 20 μg/mL.

The incubation time of the human synovial fluid with the hyaluronidaseis at least 20 minutes, preferably at least 25 minutes and evenpreferably at least 30 minutes. More preferably, the time of incubationof the human synovial fluid with the hyaluronidase is 30 minutes or 1hour. Centrifugation of the human synovial fluid sample is performedaccording to standard methods. For instance, the centrifugation can beperformed from 10,000 to 15,000 rpm, such as at or about 13,000 rpm for5 to 15 minutes, preferably 10 minutes.

In another aspect the invention provides a method for quantification ofa high positively charged protein in a human synovial fluid samplecomprising the steps of

-   -   a) pre-treating the human synovial fluid sample, the        pre-treating step comprising        -   adding hyaluronidase solution to the human synovial fluid            sample,        -   incubating said sample at room temperature (RT)        -   centrifuging the human synovial fluid sample    -   b) diluting the pre-treated human synovial fluid sample with a        buffer,    -   c) immobilizing a biotinylated antibody against the high        positively charged protein to a column,    -   d) washing the column to remove unbound antibody with a standard        wash buffer,    -   e) contacting in the column the pre-treated and diluted human        synovial fluid sample with the immobilized biotinylated antibody        under conditions in which the antibody binds specifically to the        high positively charged protein, to produce an antibody-protein        complex,    -   f) washing the column complex with a standard wash buffer;    -   g) adding to the antibody-protein complex in the column a        fluorescent dye labelled antibody specific for the high        positively charged protein to produce a measurable response, and        washing the column with a standard wash buffer,    -   h) measuring the response produced,    -   i) determining a quantity of high positively charged protein in        the sample by comparing the response produced with the sample to        the response produced with a calibration standard.

In further aspect the invention provides a method for automaticquantification of a high positively charged protein in a human synovialfluid sample comprising the steps of

-   -   a) pre-treating the human synovial fluid sample, the        pre-treating step comprising        -   adding hyaluronidase solution to the human synovial fluid            sample,        -   incubating said sample at room temperature (RT)        -   centrifuging the human synovial fluid sample    -   b) diluting the pre-treated human synovial fluid sample with a        buffer,    -   c) immobilizing a biotinylated antibody against the high        positively charged protein to a column,    -   d) washing the column to remove unbound antibody with a standard        wash buffer,    -   e) providing an injection means for automatic transfer of the        pre-treated and diluted human synovial fluid sample to the        column,    -   f) washing the injection means with a high ionic force buffer        before the pre-treated and diluted human synovial fluid sample        transferring to the column,    -   g) transferring the pre-treated and diluted human synovial fluid        sample to the column, thereby contacting the pre-treated and        diluted human synovial fluid sample with the immobilized        biotinylated antibody under conditions in which the antibody        binds specifically to the high positively charged protein, to        produce an antibody-protein complex,    -   h) washing the injection means with a high ionic force buffer        after the step g)    -   i) washing the column with a standard wash buffer;    -   j) adding to the antibody-protein complex in the column a        fluorescent dye labelled antibody specific for the high        positively charged protein to produce a measurable response, and        washing the column a standard wash buffer,    -   k) measuring the response produced,    -   l) determining a quantity of the high positively charged protein        in the sample by comparing the response produced with the sample        to the response produced with a calibration standard.

According to invention, the term “high positively charged protein”refers to proteins that contain more positively charged basic aminoacids (such as lysine, arginine and histidine) than negatively chargedacidic amino acids and/or are folded so that positively charged aminoacids are exposed on their surface and/or exhibit an overall positivecharge at the pH of the medium. Preferably, the high positively chargedprotein is an FGF-18 protein. More preferably, the FGF-18 protein isselected from the group consisting of: 1) a polypeptide comprising orconsisting of the mature form of human FGF-18, corresponding to thesequence comprising or consisting of residue 28(Glu) to residue 207(Ala)of SEQ ID NO: 1, 2) a polypeptide comprising or consisting of atruncated form of human FGF-18 comprising or consisting of residue 28(Glu) to residue 196 (Lys) of SEQ ID NO:1, and 3) a polypeptidecomprising or consisting of SEQ ID NO:2. More preferably, FGF-18 issprifermin.

In a further embodiment, the pre-treated human synovial fluid sample isdiluted 1:2 to 1:10, preferably at or about 1:5, with Rexxip HN buffer(above step b)).

In another embodiment the high ionic force buffer is a buffer containinga high concentration of salt such as NaCl, preferably in alcohol.Preferably the salt concentration is at least 1M. For instance, the highionic force buffer is 1.5M NaCl in 20% ethanol.

Centrifugation of the human synovial fluid sample (step a)) is performedaccording to standard methods. For instance, the centrifugation can beperformed from 10,000 to 15,000 rpm, such as at or about 13,000 rpm for5 to 15 minutes, preferably 10 minutes.

The incubation time of the human synovial fluid with the hyaluronidase(step a)) is at least 20 minutes, preferably at least 25 minutes andeven preferably at least 30 minutes. More preferably, the time ofincubation of the human synovial fluid with the hyaluronidase is 30minutes or 1 hour. The hyaluronidase concentration is ranged from 0.1 to30 μg/mL, such as 0.1, 1, 5, 10, 15, 20, 25 or 30 μg/mL. Preferably, itis ranged from 5 to 20 μg/mL. Even preferably it is at or around 20μg/mL.

In an embodiment, the standard wash buffer can be any known standardbuffer. It is for instance a Tween buffer, such as 0.05% Tween 20 inPBS.

An antibody against high positively charged protein, such as againstFGF-18 protein (i.e. an antibody that specifically binds to for exampleFGF-18 protein) can be obtain by any standard methods known in the art.

A column used in the methods of invention is any suitable column knownin the art for separating biochemical mixtures based on a highlyspecific interaction such as that between antigen and antibody.Preferably, the column is an affinity capture column. More preferably,the column is a streptavidin-bead column.

Washing steps of the column are always done to remove unbound reagents(antibodies, mixture or proteins). Washing steps of the injection meansare always done to remove sticky high positively charged protein, suchas FGF-18 protein, which can cause carry over in the run, when severalsamples have to be quantified.

An injection means used in the methods of invention is any suitableinjection device for transferring liquid samples from one vessel toanother vessel, for example for transferring the pre-treated and dilutedhuman synovial fluid sample from dilution vessel to the column. Theinjection means can be disposable or non-disposable. For instance, suchsuitable injection device can be a pipetting system with a disposabletip or a needle adapted for the methods of invention. Specifically sucha needle can be a needle used in Gyrolab workstation (Gyrolabimmunoassay platform).

In the frame of automatic quantification, a set of samples can bequantified simultaneously, or nearly simultaneously. In such a case, aset of columns will be needed, one per human synovial fluid sample to beanalysed, and each step of the method will be repeated. Usually, thesame injection mean can be used to transfer the sample on the column, aslong as all the washing steps are carefully performed to avoid carryover between samples. Should a disposable injection device being used,the wash steps of steps f) and h) are not necessary.

As used herein, fluorescent dyes are organic colouring agents that areable to absorb ultraviolet radiation or visible light and emit it aslight of longer wavelength with virtually no time delay (fluorescent).Fluorescent dyes within the scope of this invention are both dyemolecules and chromophoric constituents (fluorochromes) of largermolecular units, for example chromophores bound to antibodies or otherbiomolecules. Such fluorescent dyes for example acridine dyes, cyaninedyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyesand are used in for example fluorescence analysis and as fluorescentprobes for specific labelling in immunology. A good overview of commonfluorescent dyes and their fields of use is known to the person skilledin the art from, for example, the Handbook of Fluorescent Probes andResearch Chemicals, Richard P. Haugland, Molecular Probes.

In a preferred embodiment, Alexa-647 fluorescent dye is used. Thisfluorescent dye belongs to the Alexa Fluor family of fluorescent dyesand is produced by Molecular Probes, Inc. The excitation and emissionspectra of the Alexa Fluor series cover the visible spectrum and extendinto the infrared. The individual members of the family are numberedaccording roughly to their excitation maxima (in nm). Alexa Fluor dyesare synthesized through sulfonation of coumarin, rhodamine, xanthene(such as fluorescein), and cyanine dyes. Sulfonation makes Alexa Fluordyes negatively charged and hydrophilic. Alexa Fluor dyes are generallymore stable, brighter, and less pH-sensitive than common dyes (e.g.fluorescein, rhodamine) of comparable excitation and emission, and tosome extent the newer cyanine series.

In the context of the present invention, a measurable response isfluorescence emitted by labelled compounds (antibody or protein) andmeasured by any fluorometry or spectrofluorometry methods known in theart. When Gyrolab technology is used, the fluorescence signal emitted ismeasured by the gyrolab workstation, which is able to perform a scanningof the column surface, giving as a result a numeric response of RelativeFluoresce Unit, and an image of the peak of fluorescence into thecolumn.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications without departing fromthe spirit or essential characteristics thereof. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.The present disclosure is therefore to be considered as in all aspectsillustrated and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, exemplary ofmethods of practising the present invention and are not intended tolimit the scope of the invention.

Description of the Sequences:

SEQ ID NO. 1: Amino acid sequence of the native human FGF-18.

SEQ ID NO. 2: Amino acid sequence of the recombinant truncated FGF-18(trFGF-18).

Examples

In the following examples, if an actual sample concentration is belowthe lower limit of quantitation of the method (150 μg/mL), it will bereported as below lower limit of quantitation (BQL).

1. Affinity Measurement Using Gyrolab Technology 1.1.1. Analysis Method

For the affinity measurement a direct antibody assay was used. Theantigen, Sprifermin, labelled with biotin, is attached to the CD column,and the alexa647-labelled mAb is captured by the immobilized antigen. Apre-mixing step of the antigen and antibody, performed outside the CD,in a microplate well is required. After a defined time the mixture isloaded, and only the free antibody is captured by the immobilizedantigen. The analysed antibodies are monoclonal antibodies (IgG)produced against Sprifermin by Zymogenetics and used for thecre-clinical and clinical PK assay. The antibodies are identified asF5A2 (referred in the text as mAb F05) and F44A2 (referred in the textas mAb F44).

1.1.2. Sprifermin Biotinilation

For biotinilation the kit Pierce “EZ-Link MicroSulfo-NHS-LC-Biotinylation: no-weight biotin” (cat. 21327) was used. Thebiotinilated product was purified using Pierce “Zeba Desalt SpinColumns” (cat. 89849), while the degree of labelling was checked byusing the kit from Pierce “Biotin quantitation kit” (cat. 25008).N-Hydroxysuccinimide (NHS) esters of biotin are the most popular type ofbiotinylation reagents. NHS-activated biotins react efficiently withprimary amino groups (—NH2) in pH 7-9 buffers to form stable amidebonds. Proteins typically have many sites for labelling (see FIG. 1),including the primary amine in the side chain of lysine (K) residues andthe N-terminus of each polypeptide. Several different NHS esters ofbiotin are available with varying properties and spacer arm lengths. Thesulfo-NHS ester reagent in this kit is water-soluble, enabling reactionsto be performed in the absence of organic solvents. Because biotin issmall (244 Da), it can be conjugated to many proteins without alteringtheir biological activities.

The biotination was performed according to this procedure:

-   -   1. 200 μL of cold Milli-Q water were added to 1 mg of biotin to        obtain a concentration of 9 mM;    -   2. The Sprifermin was brought to a concentration of 0.1 mM        (corresponding to 2 mg/mL: molecular weight 19830). A 2/10/20        fold molar excess of biotin over the analyte is used. Therefore,        2.7/13.5/27 μl of 9 mM biotin is added to 120 μl Sprifermin;    -   3. The mixture was incubated for 1 h in shaking condition;    -   4. Excess of biotin was removed with a Zeba Desalt Spin Column        from Pierce. This column is able to exchange buffer in which the        protein is dissolved with ≧95% retention of salts and small        molecules while providing recovery of proteins greater than        7,000 Da. First a column was centrifuged for 1 minute at 1500×g.        The excess of liquid was removed and 120 μL of sample was        loaded. The column was centrifuged for 2 minutes at 1500×g. The        flowthrough contains the labelled protein without free biotin.    -   5. The sample concentration was measured by spectrometry by        reading at 280 nm with Nanodrop ND-1000. PBS was used as        reference.

After biotinilation the labelling degree was checked by using the PierceBiotin Quantitation Kit. HABA (4″-hydroxyazobenzene-2-carboxylic acid)is a reagent that enables a quick estimation of the mole-to-mole ratioof biotin to protein. The kit contains a premix of HABA and avidin and abiotinylated horseradish peroxidase (HRP) positive control: theHABA/Avidin.

The solution containing the biotinylated protein was added to a mixtureof HABA and avidin. Because of its higher affinity for avidin, biotindisplaces the HABA and the absorbance at 500 nm decreasesproportionately. By this method, an unknown amount of biotin present ina solution can be quantified in a single microplate well by measuringthe absorbance of the HABA-avidin solution before and after addition ofthe biotin-containing sample. The change in absorbance relates to theamount of biotin in the sample by the extinction coefficient of theHABA-avidin complex.

The following procedure was applied:

-   -   1. No-Weigh HABA/Avidin Premix was equilibrated to room        temperature and dissolved in 100 μL of milliQ water;    -   2. 160 μl of PBS was pipetted in a microplate well for each        sample plus one for the positive control;    -   3. 20 μl of the HABA/Avidin Premix solution was added to the PBS        in each well. The microplate was placed on an orbital shaker to        mix for 2 minutes;    -   4. Absorbance of the solution in the well at 500 nm was measured        and the value was recorded as A500 HABA/avidin;    -   5. 20 μl of biotinylated sample/Biotinylated HRP (positive        control with know biotinilation rate) were added to the well        containing the HABA/avidin reaction mixture and mixed as        described above;    -   6. The absorbance of the solution in the well was read at 500 nm        and the value was recorded as A500 HABA/avidin/biotin sample        once the value remains constant for at least 15 seconds;    -   7. Finally the milliMoles of Biotin per milliMole of Protein (in        the original sample) was calculated according to the following        formulas:

${{mmol}\mspace{14mu} {of}\mspace{14mu} {biotinilated}\mspace{14mu} {protein}\mspace{14mu} {per}\mspace{14mu} {mL}} = {\frac{{protein}\mspace{14mu} {{conc}.\mspace{14mu} \left( {{mg}\text{/}{mL}} \right)}}{{MW}\mspace{14mu} {of}\mspace{14mu} {protein}\mspace{14mu} \left( {{mg}\text{/}{mmol}} \right)} = {{Calc}\mspace{14mu} {\# 1}}}$  Δ A₅₀₀ = (A₅₀₀H ∖ A) − (A₅₀₀H ∖ A ∖ B) = Calc  #2$\frac{{mmol}\mspace{14mu} {biotin}}{{mL}\mspace{14mu} {reaction}\mspace{14mu} {mixture}} = {\frac{\Delta \; A_{500}}{34000\left( {{light}\mspace{14mu} {path}\mspace{14mu} {lenght}\mspace{14mu} {cm}} \right)} = {{Calc}\mspace{14mu} {\# 3}}}$$\mspace{20mu} {\frac{{mmol}\mspace{14mu} {biotin}}{{mmol}\mspace{14mu} {protein}} = \frac{{\left( {{Calc}\mspace{14mu} {\# 3}} \right)10{dilution}}\mspace{14mu} {factor}}{{Calc}\mspace{14mu} {\# 1}}}$

1.1.3 Alexa-Fluor 647 Labelling of the Antibodies

The kit from Molecular Probes “AlexaFluor® 647 Monoclonal AntibodyLabeling Kit” (cat. A20186) was used to label and purify the product.Nanodrop 1000 was used to check the degree of labelling. Conjugates haveabsorption and fluorescence maxima of approximately 650 nm and 668 nm,respectively. The Alexa Fluor 647 reactive dye has a succinimidyl estermoiety that reacts efficiently with primary amines of proteins to formstable dye-protein conjugates.

The labelling was performed according to this procedure:

-   -   1. The antibodies were diluted to the concentration of 1 mg/mL,        and then one-tenth volume of Sodium Bicarbonate buffer (pH 8.3)        was added. (Bicarbonate, pH˜8.3, is added to raise the pH of the        reaction mixture, since succinimidyl esters react efficiently at        pH 7.5-8.5);    -   2. 100 μL of the protein solution (from step 1) was transferred        to the vial containing the reactive dye, and gently inverted to        fully dissolve the dye;    -   3. The solution was incubated for 1 hour at 22° C. in shaking;    -   4. In order to separate the labelled antibody from the unreacted        product, excess of dye was removed using a 30,000 MW size        exclusion resin in phosphate-buffered saline (PBS), pH 7.2, plus        2 mM sodium azide. A spin column was placed in a 5 mL tube and        the purification resin was stirred. 1 mL of the suspension was        added into the column and allowed to settle. Then 500 μL were        added. The total suspension volume is 1.5 mL. The column buffer        was allowed to drain from the column by gravity. Initially, some        pressure may be required to cause the first few drops of buffer        to elute;    -   5. The spin column was placed in one collection tube and        centrifuged for 3 minutes at 1100×g using a swinging bucket        rotor. The buffer was discarded. The spin column is now ready        for purifying the conjugated antibody;    -   6. The product from step 3 (labelling mixture) was added drop        wise onto the centre of the spin column and allowed to absorb        into the gel bed;    -   7. The spin column was place into the empty collection tube and        centrifuged for 5 minutes at 1100×g;    -   8. After centrifugation, approximately 100 μL of labelled        protein in PBS, pH 7.2, with 2 mM sodium azide was collected.        Free dye remained in the column bed.

To quantitate the labelling ratio, the absorbance of the conjugatesolution was read at both 280 nm and 650 nm using the Nanodropinstrument. The concentration of protein in the sample was calculatedaccording to the following formula:

${{Protein}\mspace{14mu} {concentration}\mspace{14mu} (M)} = \frac{\left\lbrack {A_{280} - {{\left( {A_{650}0.03} \right){Dilution}}\mspace{14mu} {Factor}}} \right\rbrack}{{203'}000}$

where 203'000 is the molar extinction coefficient (ε) in cm⁻¹M⁻¹ of atypical IgG at 280 nm, and 0.03 is a correction factor for thefluorophore's contribution to the absorbance at 280 nm.

The degree of labelling was calculated in this way:

${{Moles}\mspace{14mu} {of}\mspace{14mu} {dye}\mspace{14mu} {per}\mspace{14mu} {moles}\mspace{14mu} {of}\mspace{14mu} {protein}} = \frac{{A_{650}{Dilution}}\mspace{14mu} {Factor}}{{239'}{000{Protein}}\mspace{14mu} {concentration}\mspace{14mu} (M)}$

where 239'000 cm⁻¹M⁻¹ is the approximate molar extinction coefficient ofthe Alexa Fluor 647 dye at 650 nm. For IgGs, the optimal labelling isbetween 3 and 7 moles of Alexa Fluor 647 dye per mole of antibody.

1.1.4. Capture Reagent Investigation

In order to determine the best experimental condition for the capturereagent (biotinilated Sprifermin), different conditions were tested in a2-step Gyrolab assay. The biotinylated capturing reagent is bound to thebeads creating analyte-specific capture columns. During the assayprocess, the alexa-647 labelled antibodies are passed through thecolumns, where the antibody is specifically captured.

The following capturing reagents were used:

-   -   Unlabelled Sprifermin alone;    -   Unlabelled Sprifermin+Biotinilated Bovine Serum Albumin        (Bio-BSA, Sigma, A8549) ratio 1:3;    -   Unlabelled Sprifermin+Bio-BSA ratio 1:9;    -   Sprifermin, biotinilation labelling 1:2;    -   Sprifermin, biotinilation labelling 1:10;    -   Sprifermin, biotinilation labelling 1:20;

All the capturing reagents were brought to the concentration of 0.1mg/mL in PBS-T (0.01% Tween 20 in Phosphate Buffered Saline) or inRexxip HN buffer (Gyros, P0004996). The detection reagent (alexa-647labelled mAb anti-sprifermin) was tested at the concentrations of 5, 10and 50 nM diluted in Rexxip F buffer (Gyros, P0004825). The firstexperiment was performed using the Gyrolab method “Bioaffy 1000 2 stepC-AD wiz-v1”. The second experiment was performed using the Gyrolabmethod “Bioaffy 1000 wash ×2 C-AD wiz-v1” with the addition of extrawashing steps using Wash Solution 2 (WS2: 1.5M NaCl in 20% Ethanol).

Results

The first experiment to assess the best capture condition was performedby comparing three different degrees of biotinilated Sprifermin (ratioSprifermin:biotin 1:2, 1:10, 1:20), and unlabelled Sprifermin as anegative control. The detection mAb under evaluation was prepared atthree different concentrations. The results of the combination of thedifferent capture conditions showed that there's a high unspecificsignal due to the binding of the drug to the streptavidine-capturedcolumn (See Table 1, Sprifermin unlabelled).

TABLE 1 Results for the capture reagent investigation. First Experiment.Sprifermin Sprifermin Sprifermin Alexa 647 biotinilation biotinilationbiotinilation Sprifermin mAb (conc. degree 1:2 degree 1:10 degree 1:20unlabelled nM) AVG RLU AVG RLU AVG RLU AVG RLU 0 0.237 0.012 0.033 0.0065 678.3 71.7 11.8 72.6 10 686.7 80.9 13.3 106.2 50 710.3 135.4 16.5150.3 AVG RLU: average response of three replicates.

In order to gain specificity, a change in the gyrolab method was done. Awashing step with a high ionic force buffer (WS2) was added to reducethe non-specific binding. The unlabelled Sprifermin was tested also incombination with Bio-BSA in order to block free binding sites of thecolumn. Results of the second experiment are shown in Table 2.

TABLE 2 Results for the capture reagent investigation. SecondExperiment. Sprifermin Sprifermin Sprifermin Alexa 647 biotinilationbiotinilation biotinilation mAb (conc. degree 1:2 degree 1:10 degree1:20 nM) AVG RLU AVG RLU AVG RLU 0 0.008 0.032 −0.012 5 330.5 402.0 1.910 308.0 485.0 2.2 50 751.7 534.0 2.7 Sprifermin Sprifermin Alexa 647Sprifermin unlabelled + unlabelled + mAb (conc. unlabelled bio-BSA 1:3bio-BSA 1:9 nM) AVG RLU AVG RLU AVG RLU 0 0.005 −0.004 0.002 5 5.8 6.24.3 10 9.6 11.4 5.8 50 14.4 20.0 8.5 AVG RLU: average response of threereplicates

Using the new method, with WS2 washing steps, the specific signal due tothe binding of bio-Sprifermin to the column is higher in relation to thesignal of the unlabelled drug (at alexa labelled mAb concentration of 50nM, with the first method the ratio between Sprifermin labelled 1:2 andSprifermin unlabelled is around 5. In the second experiment the ratio isaround 50). The responses of the unlabelled drug drastically decreasefrom experiment 1 to experiment 2, while the response of the labelleddrug remains constant. The addition of Bio-BSA doesn't seem to helpsignificantly the reduction of a specific signal. By analysing the peaksof the different conditions, there's a clear difference among the signalgenerated by using labeled and unlabelled Sprifermin. The mostinteresting condition, in terms of peak shape and signal saturation,seems to be the Sprifermin biotinilation degree 1:10.

1.1.5. Fixed Antibody (Ab) Concentration Determination

In order to choose the fixed Alexa-647 labelled mAb concentration forthe K_(D) experiments, a standard curve spanning differentconcentrations of Alexa-647 labelled MAb was prepared withoutpre-incubation with the antigen. The different Ab concentrations weretested in a 2-step Gyrolab assay “Bioaffy 1000 wash ×2 C-AD wiz-v1”. Thecapture reagent, Bio-Sprifermin, labelling degree 1:10 was used at 0.1mg/mL diluted in Rexxip HN. The standard curve was prepared by dilutingAlexa-labelled mAb F05 at 50, 1, 0.1, 0.01, 0.005, 0.001 nM, 0.5 and 0.1pM in Rexxip F. And Alexa-labelled mAb F44 at 50, 10, 1, 0.01, 0.005,0.001 nM and 0.5 and 0.1 pM in Rexxip F.

Results

In order to establish the fixed mAbs concentrations to be used for theK_(D) experiments, the two antibodies were prepared at differentconcentrations without pre-incubation with the drug. After a couple ofexperiments to determine the linear part of the sigmoidal curve, itseems that below 0.01 nM the responses are not stable for bothantibodies showing quite similar behaviours (see Table 3). The % CVresulted very high up to 0.01 nM. The chosen concentration was 0.1 nMfor both antibodies.

TABLE 3 Results for the F05 and F44 Fixed Ab concentration determinationexperiment. Ab Conc. Alexa 647 lab. mAb F05 Alexa 647 lab. mAb F44 (nM)AVG RLU % CV AVG RLU % CV 0 1.687 140.3 2.601 88.9 0.0001 0.812 135.01.150 86.4 0.0005 0.456 121.1 1.077 77.4 0.001 0.355 107.8 1.226 64.10.005 0.616 54.7 2.217 37.8 0.01 1.1 22.1 3.9 36.2 0.1 8.8 2.3 40.0 4.71 69.5 1.9 483.0 7.9 10 n.a. n.a. 735.0 1.9 AVG RLU: average response ofthree replicates, CV %: % coefficient of variation of three replicates.In red: % CV out of the acceptance criteria (±20%)

1.1.6. Equilibrium Time Determination and Evaluation Ration [Ab]/K_(D)

In order to choose the correct incubation time for the K_(D)experiments, a standard curve spanning different concentrations ofunlabelled Sprifermin was prepared and then incubated with the fixedalexa-647 labelled MAb for 1 h or 24 h and then tested. The standardcurves were tested in a 2-step Gyrolab assay “Bioaffy 1000 wash ×2 C-ADwiz-v1”. The capture reagent, Bio-Sprifermin, labelling degree 1:10, wasused at 0.1 mg/mL diluted in Rexxip HN. The standard curve was preparedby diluting unlabelled Sprifermin at a final concentration of 0.0005,0.01, 0.05, 0.25, 0.5, 5, 50, 500, 1000 and 2000 nM. The dilution was a1:20 performed in an Alexa-labelled mAb F05 at 0.1 nM, in Rexxip F. AndAlexa-labelled mAb F44 at 0.1 or 0.05 nM in Rexxip F. These mixes werekept in shaking, in the dark for 1 h or 24 h at 22° C.

Results

The chosen mAb concentration of 0.1 nM was pre-incubated with varyingconcentrations of Sprifermin in order to choose the preferred incubationtime for K_(D) determination. The pre-incubation was performed for 1 hor 24 h and then tested in order to assess if the shorter incubation wasable to allow the equilibrium of the reaction (see FIG. 2 for the mAbF05 results).

For the mAb F44 stable results were more difficult to obtain, thereforeit was decided to decrease the fixed mAb concentration from 0.1 to 0.05nM. The results in terms of % BIAS were not optimal, but were confirmedin many experiments. Since the calculated K_(D) resulted to be similarafter 1 and 24 h incubation, it was decided to keep the incubation timeat 1 h (see FIG. 3 for the mAb F44). For both antibodies the ratio[Ab]/K_(D) resulted to be abundantly below 1, so the experimentperformed can be called K_(D) controlled, allowing an accuratedetermination of the K_(D).

1.1.7. Confirmation of the K_(D)

In order to confirm the obtained data, the determination of the K_(D)was repeated 4 times for each antibody following the same procedure seenin 1.1.6.

Results

KD experiments were repeated 4 times for each antibody in order toconfirm the results. See FIG. 4 for mAb F05, see FIG. 5 for mAb F44. F05gave robust results, the calculated K_(D) is conserved in all theexperiments and responses are stable. The calculated K_(D) resulted tobe around 1 nM. F44 resulted to be less reproducible, probably thereason is related to the low concentration of reagents used. mAb F44shows however an higher affinity for Sprifermin than mAb F05, since thecalculated K_(D) is around 300 pM, and the high variation in theinstrumental responses can be ascribed to the fact that this highaffinity is probably a challenge for this technology.

1.1.8. KD Determination at Different Ab Fixed Concentrations

To establish the goodness of the calculated K_(D)s, unlabelledSprifermin was incubated with various mAb concentrations. A standardcurve spanning different concentrations of unlabelled Sprifermin wasprepared and then incubated with 4 fixed alexa-647 labelled MAb for 1 h.The standard curves were tested in a 2-step Gyrolab assay “Bioaffy 1000wash ×2 C-AD wiz-v1”.

The capture reagent, Bio-Sprifermin, labeling degree 1:10 was used at0.1 mg/mL diluted in Rexxip HN. The standard curve was prepared bydiluting unlabelled Sprifermin at a final concentration of 0.0005, 0.01,0.05, 0.25, 0.5, 5, 50, 500, 1000 and 2000 nM. The dilution was a 1:20performed in an Alexa-labelled mAb F05 at 0.01, 0.1, 1 and 10 nM, inRexxip F. And Alexa-labelled mAb F44 at 5, 50 pM, 0.5 and 5 nM in RexxipF. These dilutions were kept in shaking, in the dark for 1 h at 22° C.

Results

To confirm the reliability of the calculated K_(D), the two mAbs wereincubated at four different concentrations with unlabelled Sprifermin(as in previous experiments). This experiment was done in order toexclude that the obtained K_(D) could be valid only for the chosen Abconcentration used in all the previous experiments. For the mAb F05,variations in the concentration had low impact on the calculated K_(D).Only the highest concentration tested: 10 nM (100 times higher than thechosen 0.1 nM) showed a different result, but since the [Ab]/K_(D) is1.6 this determination can't be considered as accurate as the others(data not shown). The same happened also for the mAb F44 (data notshown). However the K_(D) resulted to be higher than the one obtained inthe previous experiment, confirming the hypothesis that the higheraffinity of this antibody for its antigen is challenging for the Gyrolabtechnology.

2. Epitope Mapping Using Pepscan CLIPS Technology

The Pepscan CLIPS technology was used for the characterization of thetwo mAbs (F44 and F05) used for the PK immunoassay to quantifySprifermin in serum and synovial fluid. The target drug was converted ina library of around 4000 overlapping peptide constructs, using acombinatorial matrix design. The CLIPS technology allows to structurepeptides into single, double or triple loops. In this case single anddouble loops were tested. The synthesis was performed coupling CLIPStemplates to cysteine residues in the peptides. Binding of theantibodies to each peptide was tested using a PEPSCAN-based ELISA. Thepeptide arrays were incubated with primary antibody solution overnightat 4° C. After washing, the peptide arrays were incubated with a 1:1000dilution of an antibody peroxidase conjugate (SBA, cat. 2010-05) for 1hour at 25° C. After washing, the peroxidase substrate2,2′-azino-di-3-ethylbenzthiazoline (ABTS) and 2 μL/mL of 3% H₂O₂ wereadded. Colour development was measured after one hour and quantifiedwith a CCD camera and an imaging processing system.

Six different sets of peptides were synthesized:

-   -   Set 1: Single residue mutagenesis scan: systematic replacement        of a single position of a base sequence with any of the 19        natural amino acids (about 300 peptides);    -   Set 2: CLIPS conformational loops: Single-loop conformational        CLIPS peptides, complete set of 20mers with an overlap of 19        residues (about 150 peptides);    -   Set 3: CLIPS conformational loops with mismatch disruptions:        Identycal to set 2, but the 2 amino acids in the central        position are substituted with Ala (about 150 peptides);        Comparing Set 2 and 3 results can indicate the relevance of a        mutated position.    -   Set 4: Linear: linear 20mers overlapping for 19 residues. (About        150 peptides);    -   Set 5: Linear with mismatch disruptions: Identycal to set 4, but        the 2 amino acids in the central position are substituted with        Ala (about 150 peptides);    -   Set 6: CLIPS discontinuous matrix: a matrix of 26×154 17mers        with an overlap of 16 residues. It is modelled on T3 CLIPS        scaffold (about 4000 peptides);

In order to optimize the binding conditions, an experimental design wasperformed with varying concentrations of the antibody, the compositionof the diluent buffer and the array pre-treatment.

Results

In order to highlight the best binding peptides, a statistical tool thatcan be used is the box-plot method. The distribution of the resultsshows a common background level, and enables the determination ofstatistical outliers: peptides with a statistically relevant higherbinding among the noise, which could indicate potential bindingpeptides. Using the box-plot analysis, the best conditions to highlightspecific binding resulted to be antibody concentration at 5 μg/mL with1% of competitive proteins in the blocking buffer.

Analysis of the linear and CLIPS peptides indicates that both antibodieshave a similar binding pattern, with higher binding at the N- andC-term. Analysis of the discontinuous CLIPS matrix showed specificbinding patterns for the 2 antibodies. The following amino acidsnumbering is based on SEQ ID NO. 2. For both F05 and F44 the observeddominant binding regions were 21-27 and 36-45. Binding by F44 was alsoobserved on regions 121-129, 141-151 and 153-163. These two regions alsoshowed some binding for F05, but to a much lesser extent. F05 was shownto also bind to the N-terminal 1-21.

In order to clarify binding differences among the two antibodies, thevalues from this data set were compared: each response value was plottedin a pair wise scatter plot. If for one peptide the binding is higherfor one antibody in respect to the other, this point cloud would not becentred on a 1:1 line. As overall binding for F44 is higher than forF05, the point cloud in fact is not centred on a 1:1 line (see FIG. 6A).To adjust for overall binding and focus on specific differences betweenthe two samples, the point cloud was normalized using a LOESS curve fit(see FIG. 6B). After normalization, the point cloud is centred around1:1. To identify differentially binding peptides, the data from FIG. 6Bis re-fitted into an MA-plot (a plot type which shows the mean value oftwo samples onto the X-axis, and the log 2 of the difference on the Yaxis). On this plot, differentially binding peptides are those with ahigh Y-value. To remove noise, a cutoff for both mean and differentialbinding can be applied. The remaining differentially binding peptidesare coloured blue (higher binding in F44) and yellow (higher binding inF05) in FIG. 6C. The identified 218 peptides (out of 4000) were scoredaccording to sequence positions on the target protein (FIG. 6D). A clearincreased binding for F44 is observed for peptides covering region153-164. For F05, high relative binding is mainly observed for 7-21.

Both antibodies show strongest binding to region 21-27, suggesting thisregion is the epitope for both F05 and F44. However, secondary evidenceshows that the two antibodies have different epitopes, as they can beused together in a ‘sandwich-ELISA’ experiment. Differential analysisbetween F05 and F44 suggests specific binding by F05 on the N-terminaldomain and binding by F44 on the C-terminal domain. Both these domainsare in vicinity of the dominant binding region. These results can bevisualized as in FIG. 7, by using a protein belonging to the same familyof Sprifermin.

The putative epitopes for F05 and F44 are therefore:

-   -   F05: 21-27 together with 7-21    -   F44: 21-27 together with 153-164        3. Development of a Gyrolab Based Assay for PK        (Pharmaco-Kinetics) with Analyte and Matrix

3.1. Search for the Best Antibody Combination

In order to determine the best antibody couple combination two differentconditions were tested in a 3-step Gyrolab assay: “Bioaffy 1000 wiz v1mod wash” with the addition of Wash Solution 2 (WS2: 1.5M NaCl in 20%Ethanol). A standard (std) curve of the analyte, Sprifermin, wasprepared with independent dilutions in Rexxip HN buffer (Gyros,P0004996) at 50, 10, 2 ng/mL, 400, 80, 16, 3.2 μg/mL. A blank sample wasadded (Standard 0, “STD0”, only Rexxip HN). The capture reagents (F44and F05 antibodies, biotin labelled) were used at the concentration of0.1 mg/mL. The detection reagent (alexa-647 labelled F44 and F05) wereused at the concentration of 25 nM diluted in Rexxip F buffer (Gyros,P0004825).

Results

To decide the optimal antibody combination for this PK assay, thecombinations of the two mAbs (F05 as capture/F44 as detection and F44 ascapture/F05 as detection) were tested using a 3 step

Gyrolab method “Bioaffy 1000 wiz v1 mod wash” with the addition of WashSolution 2 (see FIG. 8). This experiment was repeated 3 times, also withvariations in the detection antibody concentrations (data not shown).The best combination resulted to be the one with F44 as capture and F05as detection: this binding arrangement produced peaks with a sharpershape and it showed to allow a better linearity also in the lower partof the curve (data not shown).

3.2. Choice of the Buffer

The choice of the buffer for the analyte dilution is crucial for theperformance of the immunoassay. Four different buffers were used for thepreparation of a standard curve of Sprifermin and analyzed according tothe AlphaLISA technology procedure:

-   -   Rexxip HN buffer (Gyros, P0004996);    -   Rexxip HN MAX buffer (Gyros, P0004997);    -   Diluent buffer 1 (0.1% Bovine Serum Albumin (Sigma, cat. number        A7906), 0.05% Lutrol (BASF cat. Number S30101) in 7 mM Na2HPO4,        1 mM KH2PO4, 2.7 mM KCl, pH 7.3);    -   Diluent buffer 10 (1% Bovine Serum Albumin (Sigma, cat. number        A7906), 0.5% Lutrol (BASF cat. Number S30101) in 7 mM Na2HPO4, 1        mM KH2PO4, 2.7 mM KCl, pH 7.3).

STD curve was prepared at 20, 5, 1 ng/mL, 200, 50, 25, 10 μg/mL (finalconcentration in the well). A blank sample was added (STD0, onlybuffer). The capture reagent, F44 biotin labeled, was used at theconcentration of 0.1 mg/mL. The detection reagent, alexa-647 labelledF05, was used at the concentration of 25 nM diluted in Rexxip F buffer(Gyros, P0004825).

Results

The best buffer resulted to be Rexxip HN, a commercial reagentcontaining agents to neutralize heterophilic antibodies, with anincreased ionic strength for positively charged analytes, as Spriferminis. The standard curve prepared in Rexxip HN had better accuracy (seeTable 5) and precision (see Table 4) towards the other curves.

TABLE 4 Resuming table of the experiment for the buffer choice.Sprifermin nominal Rexxip HN DB 10 DB 1 Rexxip HN MAX concentration AVGAVG AVG AVG pg/mL RLU % CV RLU % CV RLU % CV RLU % CV 0 0.191 1.1 0.13624.6 0.110 20.3 0.163 36.8 10 0.414 3.6 0.372 124.3  0.129 30.4 0.490 3.0 25 0.637 8.9 0.131 26.2 0.237 77.0 1.091 11.6 50 1.16 11.7 0.18 5.7 0.20 19.0 2.16  9.6 200 4.65 17.7 0.44 27.9 0.61 17.7 3.36 22.81000 20.9 5.5 2.1 30.9 2.4 13.7 16.2  9.3 5000 93.3 1.0 12.3 30.5 16.7 5.7 63.4  7.1 20000 329 2.6 67 34.0 84  7.4 237 11.9 AVG RLU: averageresponse of three replicates, % CV: precision of three replicates.Underlined: % CV out of the acceptance criteria (±20%)

TABLE 5 Resuming table of the experiment for the buffer choice.Sprifermin Rexxip HN DB 10 DB 1 Rexxip HN MAX nominal AVG AVG AVG AVGconcentration BCC % BCC % BCC % BCC % pg/mL pg/mL BIAS pg/mL BIAS pg/mLBIAS pg/mL BIAS 10 11.2 12.0 253.5 2434.5   32.5 225.0  7.6 −24.4  2523.4 −6.5 30.4 21.6  71.4 185.5  36.6 46.4 50 51.5 3.1 65.2 30.4  56.613.1 95.6 91.2 200 207.5 3.8 217.0 8.5 242.0 21.0 168.7 −15.7  10001043.7 4.4 1052.3 5.2 904.7 −9.5 1059.3  5.9 5000 4783.3 −4.3 5423.3 8.55080.0  1.6 4963.3 −0.7 20000 20366.7 1.8 16100.0 −19.5  20250.0  1.320050.0  0.3 AVG BCC: average back calculated concentration of threereplicates, % BIAS: accuracy of BCC. Standard curve regressions wereobtained using Xlfit (IDBS) logistic autoestimate weighted 1/y². formula201. Underlined: % BIAS out of the acceptance criteria (±20%)

3.3. Minimal Required Dilution (MRD)

Once the method has been optimized in buffer, it was translated inmatrix. For this purpose an experiment was performed following theGyrolab technology procedure. The standard curve prepared in Rexxip HNwas used to quantify the analyte prepared in Pooled human Synovial fluid(Sera Laboratories International) at the following concentrations: 0.5,1, 2, 5 and 10 ng/mL and then diluted respectively 1:5, 1:10, 1:20,1:50, 1:100. In parallel blank matrix was were treated in the same waybefore testing.

Results

In order to set the MRD to be applied for samples testing, samplesprepared in pooled human synovial fluid were analysed against astandard-curve prepared in Rexxip HN. Performing the first experimentusing synovial fluid, the matrix resulted to be very viscous anddifficult to pipette, moreover, due to the high viscosity it could alsocause needle clotting. For these reasons the result of the experimentsperformed to find the MRD were not satisfying in terms of accuracy andprecision: the % CV was high for the 1:10 dilution (see Table 6 and FIG.9), and the % BIAS was acceptable for lower MRD (1:5 and 1:10) but notfor high dilutions. For this reason, it was decided to improve thehandling of synovial fluid by adding a step of pre-treatment of thematrix before the MRD

TABLE 6 Resuming table of the experiment for the MRD. MRD 1:5 MRD 1:10MRD 1:20 MRD 1:50 MRD 1:100 Spiked SF pool (final concentration 100pg/mL) (Acc. Criteria: % BIAS ± 20%) AVG BCC pg/mL 112.0  115.9  128.7 123.7  127.7  % BIAS 12.0 15.9 28.7 23.7 27.7 % CV  5.4 25.6  2.5  1.2 7.1 Blank SF pool, acceptance criteria: all BQL replica 1 BCC pg/mL BQL13.4 44.8 BQL 11   replica 2 BCC pg/mL 10   10.8 30.1 BQL BQL replica 3BCC pg/mL BQL 11.6 34.5 BQL BQL AVG BCC: average back calculatedconcentration of three replicates, % BIAS: accuracy of BCC. % CV:precision of three replicates. BQL = Below low limit of quantification.Standard curve regression was obtained using Xlfit (IDBS) logisticautoestimate weighted 1/y². formula 201. Underlined: results out of theacceptance criteria

3.4. Matrix Pre-Treatment

In order to reduce matrix viscosity a centrifugation step and adigestion with hyaluronidase were setup.

3.4.1. Hyaluronidase Digestion

A stock solution of Hyaluronidase (Hyaluronidase Type from Bovine TestesSigma, H3506) was diluted in PBS containing 0.01% BSA at theconcentration of 200 and 2 μg/mL. These solutions were added to threesamples: 1) blank synovial fluid, 2) synovial fluid with the addition ofthe analyte at 1000 μg/mL (final concentration 100 μg/mL), and 3)synovial fluid with the addition of the analyte at 200 μg/mL (finalconcentration 40 μg/mL). These samples were incubated for 30 minutes atRT in shaking. At the end of the incubation the samples were diluted 1:5or 1:10 in Rexxip HN, and analyzed according to the procedure describedin paragraph 3.1.

Results

As shown in Table 7, an improvement of the results by digesting synovialfluid with hyaluronidase is obtained only for the 1:5 dilution both forblank and spiked samples. Among the two concentrations of Hyaluronidasetested, the best is 10 μg/mL. This treatment produced a very low % biasfor the spiked sample, but the real improvement regards the blanksamples, almost all the treated samples resulted to be BQL, in contrastto the previous results shown (see Table 6).

TABLE 7 Resuming table of the experiment for the synovial fluiddigestion. SS 1000 pg/mL in neat SF, diluted SF blank samples diluted1:10 Acc. 1:10 (final in well conc. 100 pg/mL) Criteria: all BQL Acc.Criteria: BIAS % ±20 No Hyal. 0.1 Hyal. 10 No Hyal. 0.1 Hyal. 10treatment μg/mL μg/mL treatment μg/mL μg/mL BQL 11.4 BQL 183 150 158replica 1 BCC pg/mL BQL BQL BQL 189 153 186 replica 2 BCC pg/mL 21.5 BQLBQL 188 163 173 replica 3 BCC pg/mL   186.7   155.3   172.3 AVG BCCpg/mL   86.7   55.3   72.3 % BIAS SS 200 pg/mL in neat SF, diluted SFblank samples diluted 1:5 Acc. 1:5 (final in well conc. 40 pg/mL)Criteria: all BQL Criteria: BIAS % ±20 No Hyal. 0.1 Hyal. 10 No Hyal.0.1 Hyal. 10 treatment μg/mL μg/mL treatment μg/mL μg/mL 11   BQL BQL64.4 66.1 39.5 replica 1 BCC pg/mL 16.6 BQL BQL 65.5 46   43.4 replica 2BCC pg/mL BQL BQL BQL 55.6 BQL 42.7 replica 3 BCC pg/mL 61.8 56.1 41.9AVG BCC pg/mL 54.6 40.1 4.7 % BIAS 0.01% BSA = samples treated with nohyaluronidase, Hyal 0.1 μg/mL = samples treated with the lowerconcentration of hyaluronidase, Hyal 10 μg/mL = samples treated with thehigher concentration of hyaluronidase. AVG BCC: average back calculatedconcentration of three replicates, % BIAS: accuracy of BCC. BQL= Belowlow limit of quantification. Standard curve regression was obtainedusing Xlfit (IDBS) logistic autoestimate weighted 1/y². Formula 201.Underlined: results out of the acceptance criteria

3.4.2. Centrifugation

Three spiked samples containing the analyte were prepared using pooledhuman synovial fluid at the concentrations of 75, 2.5 and 0.15 ng/mL(final concentration in well, respectively, 15000, 500 and 30 μg/mL) andwere centrifuged twice at 13000 rpm for 5 minutes. After thecentrifugation, samples were diluted 1:5 in Rexxip HN, and analyzedusing a standard curve prepared in Rexxip HN buffer (Gyros, P0004996) at20, 10, 5, 1 ng/mL, 200, 50, 20 μg/mL (final concentrations in well). Ablank sample was added (STD0, only Rexxip HN). A blank matrix wasprocessed in the same way. The run was performed following the AlphaLISAtechnology procedure.

Results

As shown in Table 8, the centrifugation steps didn't ameliorate the %BIAS of the spiked samples analyzed after the selected MRD 1:5. All thesamples were overestimated, however, samples after centrifugation wereeasier to handle than samples only treated with hyaluronidase. For thisreason it was decided to combine the two pre-treatments.

TABLE 8 Resuming table of the experiment for the synovial fluidcentrifugation. SF SS 30 SS 500 SS 15000 BLANK pg/mL pg/mL pg/mL BQL34.1 748 ALQ replica 1 BCC pg/mL BQL 53.4 723 ALQ replica 2 BCC pg/mLBQL 44.3 931 ALQ replica 3 BCC pg/mL n.a. 43.9 800.7 n.a. AVG BCC pg/mLn.a. 46.4 60.1 n.a. % BIAS AVG BCC: average back calculatedconcentration of three replicates, % BIAS: accuracy of BCC. BQL = belowlow limit of quantification. ALQ = Above limit of quantification.Standard curve regression was obtained using Xlfit (IDBS) logisticautoestimate weighted 1/y². Formula 201.

3.4.3. Hyaluronidase Digestion+Centrifugation

Three spiked samples and a blank sample were prepared using humansynovial fluid pool at the concentrations of 75, 2.5 and 0.15 ng/mL(final concentration in well, respectively, 1500, 500 and 30 μg/mL) andwere centrifuged twice at 13000 rpm for 5 minutes. A solution of 200μg/mL of hyaluronidase in PBS containing 0.01% BSA was diluted 1:20 inthese spiked samples and incubated for 30′ at RT in shaking. At the endof the incubation the samples were diluted 1:5 in Rexxip HN, andanalyzed using a standard curve prepared in Rexxip HN buffer (Gyros,P0004996) at 20, 10, 5, 1 ng/mL, 200, 50, 20 μg/mL (final concentrationsin well). A blank sample was added (STD0, only Rexxip HN). A blankmatrix was processed in the same way. The run was performed followingthe AlphaLISA technology procedure.

Results

The combination of centrifugation plus enzymatic digestion gave betterresults for all the concentrations tested except the low SS which wasnot quantifiable (below 20 pg/mL, which was the lowest standard curvepoint; see table 9). At this point the selected pre-treatment was thefollowing:

-   -   Centrifugation of the sample twice at 13'000 rpm for 5 minutes;    -   Hyaluronidase digestion: 30′ RT in shaking with 10 μg/mL of        Hyaluronidase    -   MRD 1:5 in Rexxip HN

TABLE 9 Resuming table of the experiment for the synovial fluid doubletreatment. SF SS 30 SS 500 SS 15000 BLANK pg/mL pg/mL pg/mL BQL BQL 58518100 replica 1 BCC pg/mL BQL BQL 602 13500 replica 2 BCC pg/mL BQL BQL561 16500 replica 3 BCC pg/mL n.a. n.a. 583 16033 AVG BCC pg/mL n.a.n.a. 16.5 6.9 % BIAS AVG BCC: average back calculated concentration ofthree replicates, % BIAS: accuracy of BCC. BQL = below low limit ofquantification. Standard curve regression was obtained using Xlfit(IDBS) logistic autoestimate weighted 1/y². Formula 201.

3.4.4. Impact of the Treatment on the Analyte

Three quality controls in buffer were prepared at the concentrations of150, 1500 and 75'000 μg/mL (final concentrations respectively, 30, 300,15'000 μg/mL). Half of the preparation was diluted 1:5 in Rexxip HN, andthe other half was incubated for 30′ at 22° C. with 10 μg/mLhyaluronidase.

Results

In order to verify if sample pre-treatment could harm Sprifermin, threequality controls were analysed with or without sample pretreatment.Results indicated that the hyaluronidase digestion doesn't impact theanalyte, RLUs obtained from the treated and untreated QCs arecomparable. (see Table 10 and FIG. 10)

TABLE 10 Resuming table of the results. untreated RLU treated RLU QC-LOW1.42 1.49 1.34 1.42 1.38 1.36 QC-MEDIUM 17.7 16.7 17.9 16.5 15.5 15.8QC_HIGH 351 332 338 337 341 358 RLU of the QC samples prepared in RexxipHN at three level of concentration (QC-Low = 30 pg/mL, QC-Medium = 500pg/mL, QC-High = 15000 pg/mL) With (treated) or without (untreated)hyaluronidase digestion.

3.4.5. Matrix Pre-Treatment: Optimization and Selectivity Evaluation

The experiment to assess method selectivity was performed by using 9individual human synovial fluid samples. A stock solution ofHyaluronidase was prepared in PBS 0.01% BSA at the concentration of 400μg/mL and added (1:20 dilution) to the 9 individual synovial fluidsamples. Samples were incubated for 30 minutes at RT in shaking and thencentrifuged twice at 13000 rpm for 5 minutes. After matrix pre-treatmentSprifermin was added to each of the 9 samples at the concentration of100 μg/mL (LLOQ) and diluted 1:5 in Rexxip HN according to the AlphaLISAtechnology procedure.

Results

After having tested some individual human synovial fluid samples, it wasdecided to raise the concentration of hyaluronidase to 20 μg/mL and topostpone the centrifugation steps afterwards. This was done becausesynovial fluid from different individuals did not result clear in allcases (data not shown). As shown Table 11, 9 out of 10 samples had mean% BIAS within ±25%. The acceptance criteria for selectivity is: at least80% of individual samples spiked at the LLOQ concentration must have %BIAS of ±25%. Therefore the acceptance criteria is met.

TABLE 11 Resuming table of the results. Individual Spiked AVERAGE meanBIAS % Samples at 100 pg/mL BCC ng/mL of each conc. % CV Sample 1 22.613.0 6.3 Sample 2 20.0 −0.1 16.5 Sample 3 22.5 12.3 6.7 Sample 4 31.959.6 21.1 Sample 5 20.8 4.2 15.1 Sample 6 17.9 −10.5 4.7 Sample 7 15.5−22.3 3.9 Sample 8 17.8 −11.0 11.5 Sample 9 20.9 4.5 2.9 Sample 10 15.4−22.8 16 BCC = Back calculated concentrations of individual synovialfluid samples spiked at the LLOQ concentration (100 pg/mL, finalconcentration 20 pg/mL) average of 3 replicates. % BIAS: accuracy ofBCC. % CV = precision of BCC. In red: % CV or % bias out of theacceptance criteria (±25%)

3.5. Carryover Assessment and Gyrolab Method Optimization

In order to assess the presence of a carryover effect, a custom runusing the custom method “Bioaffy 1000 v1 mod wash” with the addition ofWash Solution 2 (WS2: 1.5M NaCl in 20% Ethanol) was performed. Thefollowing preparations were done: 1) a standard curve prepared bydiluting Sprifermin at the following concentrations: 20, 10, 5, 1 ng/mL,200, 50, 20 μg/mL in Rexxip HN; 2) QC at 20 ng/mL in Rexxip HN; 3) blanksample prepared with only Rexxip HN.

The run was set up in order to make each of the 8 needle load

-   -   1. A blank sample first;    -   2. One standard curve point;    -   3. A 20 ng/mL QC sample;    -   4. A blank sample at the end.

Capture and detection reagents were used at the concentrations alreadydefined.

Results

As the Gyrolab is a robotic system in which samples are handled using afixed set of needles, the possibility of a carryover effect must beexcluded by performing a specific assessment. For this purpose anexperiment was set up, to make each needle load a blank sample, then anhigh concentrated std and then a blank sample afterwards. All the blanksbefore and after the high concentration QC gave concentrations BQL (seeTable 12), however there was an increase in the average response betweenthe before and after, indicating the possible presence of a carryover,even if at low effect.

TABLE 12 Result of the carryover assessment. BLK before High QC BLKafter (RLU) (RLU) (RLU) Needle 1 0.118 230 0.234 0.100 236 0.223 0.715243 0.366 Needle 2 0.079 232 0.248 0.038 234 0.199 0.084 257 0.205Needle 3 0.084 231 0.332 0.068 244 0.279 0.069 244 0.305 Needle 4 0.038236 0.231 0.061 246 0.369 0.060 236 0.435 Needle 5 0.058 242 0.224 0.062254 0.188 0.063 249 0.232 Needle 6 0.081 224 0.318 0.057 233 0.361 0.083233 0.467 Needle 7 0.066 252 0.272 0.016 241 0.240 0.042 252 0.294Needle 8 0.109 254 0.468 0.091 229 0.434 0.089 220 0.452 average RLU0.10  240 0.31 % CV 137.7   4.2 29.8 RLU of blank before, high QC andblank after are reported for each needle. Averaged data are presentedfor each sample. The STD. 1 RLU at the limit of quantification was0.655. Underlined: outlier

3.5.1. Impact on LLOQ Samples

In order to assess if the slight carryover observed in the previousexperiment could have an impact on samples at the LLOQ concentrations.The same assessment was performed by substituting the blank samples withpooled synovial fluid spiked samples at the LLOQ concentration (100μg/mL, final concentration in well 20 μg/mL).

The run was set up in order to make each of the 8 needle load:

-   -   1. A LLOQ sample first;    -   2. One standard curve point;    -   3. A 20 ng/mL QC sample;    -   4. A LLOQ sample at the end.

The same method used in the previous experiment was used to test a LLOQsample before and after a high concentration sample and a modifiedmethod with only minor changes of the needle washing procedure was alsoevaluated. An additional step of needle wash with WS2 solution was addedbefore and after the step of analyte addition. Moreover, during theanalyte addition step, an additional wash was added, this modifiedmethod was called “Bioaffy 1000 wash station 2 v2 beta1”.

Results

To better understand the impact of this behaviour on samples, the sameassessment for repeated substituting the blank samples with samplescontaining the analyte at the concentration of the LLOQ. A real impacton lower concentrations samples, see Table 13, was observed confirmingthe potential presence of a carryover effect.

TABLE 13 Result of the carryover assessment. LLOQ before LLOQ after (BCCpg/mL) (BCC pg/mL) Needle 1 119.2 142.2 114.4 132.9 106.6 139.0 Needle 2117.3 134.7 115.1 132.4 132.6 119.4 Needle 3 119.2 155.0 119.8 133.6103.3 133.0 Needle 4  99.9 134.4 120.1 136.5 132.4 146.4 Needle 5 113.8132.4 113.5 122.4 115.3 131.0 Needle 6 123.2 148.8 120.1 145.9 121.8152.4 Needle 7 123.4 139.3 102.1 126.6 115.6 135.4 Needle 8 126.1 158.4 97.0 142.9 105.6 139.2 average BCC  115.73  138.09 % CV  8.1  7.0 BackCalculated Concentration (pg/mL) of LLOQ before and LLOQ after arereported for each needle. Averaged data are presented for each sample.The acceptance criteria for the concentration is between 75 and 125pg/mL. Underlined: concentration out of the acceptance criteria (100 ±25%)

TABLE 14 Result of the carryover assessment. LLOQ before LLOQ after (BCCpg/mL) (BCC pg/mL) Needle 1 115.1 101.4 105.4 109.3  86.2 110.9 Needle 2 94.5 113.5  68.9  95.0  86.9  95.6 Needle 3  99.8 126.4  97.7  97.0 97.3  98.7 Needle 4 110.5 111.8 106.1 111.4 114.6 116.7 Needle 5  97.5113.7 105.4  94.0  88.3  86.9 Needle 6 104.2 109.3 137.8 107.4 113.2114.2 Needle 7 191.7 118.6  98.2 100.7 107.0  89.2 Needle 8 120.9 121.1109.3 103.3 116.0 132.5 average BCC  107.19  107.44 % CV  21.1  10.8Back Calculated Concentration (pg/mL) of LLOQ before and LLOQ after arereported for each needle. Averaged data are presented for each sample.The acceptance criteria for the concentration is between 75 and 125pg/mL. In red: concentration out of the acceptance criteria (100 ± 25%)

On the contrary, when the needle washing procedure was improved, theimpact on the low concentrated sample was very limited (see Table 14),confirming the accuracy of the optimized method to quantify realsynovial fluid samples at very low concentrations.

3.6. Final Method 3.6.1. Summary

A biotinylated mouse monoclonal antibody against Sprifermin is used ascapture reagent (F44), and an Alexa Fluor-647 labelled monoclonalantibody against Sprifermin is used as detection reagent (F05). Thecalibration standards and quality controls are prepared in Rexxip HN.Unknown samples are analysed after pre-treatment with a minimal dilutionof 1:5 in Rexxip HN. The method range is from 150 pg/mL to 50'000 pg/mLand is further extended up to 9.6 μg/mL with dilution using Rexxip HN.Calibration curve points: 10'000, 5'000, 1000, 200, 100, 50, 30, 0pg/mL. Quality Controls: QC-H: 7'000 pg/mL, QC-M: 500 pg/mL, QC-L: 90pg/mL.

3.6.2. Unknown Samples Pretreatment:

Treat unknown samples with 20 μg/mL of Hyaluronidase solution. AddHyaluronidase to the unknown sample (e.g. 2 μL of Hyaluronidase 400μg/mL to 38 μL of unknown sample), vortex and incubate for 30 minutes at22° C. in shaking. Centrifuge the unknown samples at 13,000 rpm for 10minutes using a minispin centrifuge. Dilute treated unknown samples 1:5with Rexxip HN before analysis (e.g. 4 μL unknown sample+16 μL RexxipHN).

3.6.3. Antibodies Preparation

Coating antibody must be diluted at 0.1 mg/mL in PBS-T, if it is notready to use. Detection antibody must be diluted in Rexxip F at 20 nMand kept protected from light.

3.6.4. Procedure

1. Bring all reagents at room temperature before analysis2. Put Gyrolab in stand-by mode, turn off Gyrolab control software andthe instrument, and perform internal wash station cleaning with milliQwater3. Turn on the instrument and Gyrolab control software, initialize andprime the system with PBS-T as described in the User Guide4. Perform needle desorb as described in the User Guide using WS2 once aweek.5. Connect the WS2 solution to the Wash station 2 and prime the systemas described in the User Guide6. Create a new run using the 3-steps wizard method: “Bioaffy 1000PBTM-087_PMT 5”. Calibration standards and quality controls must beassayed in triplicate while sample in single replica.7. Add calibration standards, quality controls, unknown samples, captureand detection antibodies and wash buffer to the microplate according tothe Gyrolab control loading list. Cover with a microplate lid andprotect from light8. Load microplates and CDs as required by the instrument assistedloading procedure and start the run.

Use triplicate of each STD solution. Do not include STD-0 in theregression analysis.

Fit the standard curve with a logistic (auto-estimate, weighting factor1/Y²) equation using the instrument response of each replicate of eachcalibration standard

It is possible to dilute some unknown samples before analysis ifevidences suggest that these samples have concentrations above upperlimit of quantitation (50,000 μg/mL). Perform the dilution using RexxipHN starting from the initial samples diluted at minimal dilution (1:5).However, it should be documented.

If an actual sample concentration is below the lower limit ofquantitation of the method (150 μg/mL), it will be reported as belowlower limit of quantitation (BQL).

1-12. (canceled)
 13. A method for quantification of a high positivelycharged protein in a human synovial fluid sample comprising the steps ofa) pre-treating the human synovial fluid sample, the pre-treating stepcomprising: adding hyaluronidase solution to the human synovial fluidsample, incubating said sample at room temperature (RT), centrifugingthe human synovial fluid sample, b) diluting the pre-treated humansynovial fluid sample with a buffer, c) immobilizing a biotinylatedantibody against the high positively charged protein to a column, d)washing the column to remove unbound antibody with a standard washbuffer, e) contacting in the column the pre-treated and diluted humansynovial fluid sample with the immobilized biotinylated antibody underconditions in which the antibody binds specifically to the highpositively charged protein, to produce an antibody-protein complex, f)washing the column with a standard wash buffer, g) adding to theantibody-protein complex in the column a fluorescent dye labelledantibody specific for the high positively charged protein to produce ameasurable response, and washing the column with a standard wash buffer,h) measuring the response produced, i) determining a quantity of highpositively charged protein in the sample by comparing the responseproduced with the sample to the response produced with a calibrationstandard.
 14. The method according to claim 13, wherein the highpositively charged protein is an FGF-18 protein.
 15. The methodaccording to claim 14, wherein the FGF-18 protein is selected from thegroup consisting of: a) a polypeptide comprising or consisting of theamino acid residues 28-207 of SEQ ID NO:1, b) a polypeptide comprisingor consisting of the amino acid residues 28-196 of SEQ ID NO:1, and c) apolypeptide comprising or consisting of SEQ ID NO:2
 16. The methodaccording to claim 14, wherein the FGF-18 protein is sprifermin.
 17. Themethod according to claim 13, wherein the dilution buffer of step b) isRexxip HN.
 18. The method according to claim 13, wherein the high ionicforce buffer has a salt concentration of at least 1M.
 19. The methodaccording to claim 18, wherein the high ionic force buffer is 1.5M NaClin 20% ethanol.
 20. The method according to claim 13, wherein theincubating time of step a) is 1 h.
 21. A method for automaticquantification of a high positively charged protein in a human synovialfluid sample comprising the steps of a) pre-treating the human synovialfluid sample, the pre-treating step comprising adding hyaluronidasesolution to the human synovial fluid sample, incubating said sample atroom temperature (RT) centrifuging the human synovial fluid sample b)diluting the pre-treated human synovial fluid sample with a buffer, c)immobilizing a biotinylated antibody against the high positively chargedprotein to a column, d) washing the column to remove unbound antibodywith a standard wash buffer, e) providing an injection means forautomatic transfer of the pre-treated and diluted human synovial fluidsample to the column, f) washing the injection means with a high ionicforce buffer before the pre-treated and diluted human synovial fluidsample is transferred to the column, g) transferring the pre-treated anddiluted human synovial fluid sample to the column, thereby contactingthe pre-treated and diluted human synovial fluid sample with theimmobilized biotinylated antibody under conditions in which the antibodybinds specifically to the high positively charged protein, to produce anantibody-protein complex, h) washing the injection means with a highionic force buffer after the step g) i) washing the column with astandard wash buffer; j) adding to the antibody-protein complex in thecolumn a fluorescent dye labelled antibody specific for the highpositively charged protein to produce a measurable response, and washingthe column a standard wash buffer, k) measuring the response produced,l) determining a quantity of the high positively charged protein in thesample by comparing the response produced with the sample to theresponse produced with a calibration standard.
 22. The method accordingto claim 21, wherein the human synovial fluid sample is part of a set ofsamples.
 23. The method according to claim 21, wherein the column ofstep c) is one column in a set of columns
 24. The method according toclaim 21, wherein all the steps are repeated as often as needed toautomatically quantify a high positively charged protein in a set ofhuman synovial fluid samples to be analysed.